Abstract

Objectives This study tested whether interleukin (IL)-17A is involved in the pathogenesis of mouse myocardial ischemia/reperfusion (I/R) injury and investigated the mechanisms.

Background Inflammatory processes play a major role in myocardial I/R injury. We recently identified IL-17A as an important cytokine in inflammatory cardiovascular diseases such as atherosclerosis and viral myocarditis. However, its role in myocardial I/R injury remains unknown.

Methods The involvement of IL-17A was assessed in functional assays in mouse myocardial I/R injury by neutralization/repletion or genetic deficiency of IL-17A, and its mechanism on cardiomyocyte apoptosis and neutrophil infiltration were further studied in vivo and in vitro.

The rapid restoration of blood flow through the occluded coronary artery by mechanical or pharmacological intervention is the most effective therapy to limit infarct size and improve the clinical outcome after acute myocardial infarction (1). However, reperfusion after ischemia itself causes additional cardiomyocyte death and increases infarct size in a process called myocardial ischemia/reperfusion (I/R) injury. Potential mediators of reperfusion injury include oxidative stress and inflammation (2).

Interleukin (IL)-17A is a member of the IL-17 family, which includes six structurally related isoforms: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F (3). Multiple cell types, including CD4+ αβ T cells, γδ T cells, natural killer cells, and neutrophils, produce IL-17A (4). Interleukin-17A acts on a variety of cells through its ubiquitous receptors, IL-17RA and IL-17RC, and is a critical mediator of neutrophil recruitment and migration through the induction of granulopoiesis and the production of neutrophil chemokines, including lipopolysaccharide-induced CXC chemokine (LIX), cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein-2 (MIP-2) (5).

IL-17A plays an important role in host defense and mediates autoimmune diseases (6). We and others have reported that IL-17A is linked to the pathogenesis of several cardiovascular diseases, including atherosclerosis (7–9), hypertension, viral myocarditis, and dilated cardiomyopathy (7–12). Recently, IL-17A has also been found to contribute to brain, kidney, and intestine I/R injury (13–15), but the mechanisms involved are still largely unknown.

In this study, we investigated the expression of IL-17A in reperfused ischemic myocardium and the subsets of IL-17A–releasing cells and characterized the functional involvement of IL-17A in myocardial I/R injury. We show that IL-17A is largely produced by γδT cells and plays a pathogenic role in myocardial I/R injury by inducing cardiomyocyte apoptosis and neutrophil infiltration.

Methods

Results

IL-17A increases after myocardial I/R injury

To test the involvement of IL-17A in myocardial I/R injury, we first investigated IL-17A levels in the myocardium at different reperfusion time points after I/R. IL-17A messenger ribonucleic acid (mRNA) and protein expression increased significantly as early as 1 h after reperfusion, rose progressively until 24 h, and then began to decrease, although it remained at a high level compared with the sham group until 72 h after I/R (Online Figs. 1A and 1B).

In addition to Th17 cells, γδT cells, natural killer cells, and neutrophils were reported to secret IL-17A (4). To identify which leukocytes were responsible for the elevation of IL-17A in myocardial I/R, we used intracellular cytokine staining combined with staining for various surface markers. Over 95% of the IL-17A–secreting leukocytes were CD3+ T cells, including approximately 70% γδ T cell receptor-positive (γδTCR+) cells and 13% CD4+ cells, indicating that γδT cells rather than Th17 cells were the major source of IL-17A (Online Fig. 1C). Natural killer cells and neutrophils comprised only a minor proportion of IL-17A-secreting leukocytes. After 24 h of reperfusion, there was a marked increase in the myocardial content of γδT cells. Simultaneously, the percentage of IL-17+ γδTCR+ cells relative to γδT cells also increased (Online Tables 1 and 2). These results suggest that myocardial I/R led to an increase not only in the number of infiltrated γδT cells but also in the IL-17A production of these cells, which accounted for a large part of the elevated myocardial IL-17A.

The IL-17 family consists of 5 members, including IL-17A. Among these, IL-17F has the highest degree of homology with IL-17A and exerts similar effects but to a lesser extent (8). We therefore also examined mRNA expression of IL-17B, C, D, E, and F in hearts after I/R. The level of IL-17A mRNA was much higher than that of the other IL-17 family members, approximately 5-fold greater than that of IL-17F (Online Fig. 2).

To determine the causative role of IL-17A in the development of myocardial I/R injury, we treated the myocardial I/R mice systemically with neutralizing anti–IL-17A monoclonal antibody (mAb) or recombinant IL-17A before reperfusion. Interleukin-17A expression reached a peak at 24 h after myocardial I/R and remained elevated for 72 h; therefore, we measured infarct size at 1 day or 3 days after reperfusion. The ratio of area at risk (AAR) to left ventricle (LV) area was the same in experimental and control groups at both time points, indicating that ligature was reproducibly performed at the same level of the left anterior coronary artery. However, the ratio of infarct area to AAR was significantly lower in mice treated with anti–IL-17A mAb relative to those treated with isotype and greater in mice treated with recombinant IL-17A relative to vehicle alone at both time points (Figs. 1A and 1B). Serum cardiac troponin T (cTnT) level, an index of myocyte injury, was also significantly lower in the anti–IL-17A mAb group and higher in the recombinant interleukin (rIL)-17A group (Online Fig. 3A).

To determine the effect of IL-17A on cardiac function after I/R and to demonstrate the clinical relevance of our findings, we measured ejection fraction (EF) and fractional shortening (FS) by echocardiography. The EF and FS were both remarkably reduced at 1 day and 3 days after myocardial I/R compared with the sham group. Furthermore, treatment with anti–IL-17A mAb increased EF and FS after 1 day and 3 days reperfusion, indicating improved cardiac function. Consistent with infarct size, a marked deterioration of cardiac function, as shown by a decrease of EF and FS, was observed in the rIL-17A group (Figs. 1C and 1D). To test whether IL-17A affects hemodynamic properties, we measured left ventricular end-diastolic pressure (LVEDP) and the derivative of LV pressure (dP/dt) with a Millar catheter (SPR-671, Millar Instruments, Houston, Texas). The LV hemodynamic statuses were augmented in the anti–IL-17A mAb-treated group compared with the isotype group. Specifically, LVEDP was lower, whereas maximum and minimum dP/dt, the indexes of contractility, were increased in the anti–IL-17A mAb-treated group. Conversely, treatment with rIL-17A led to a reduction of dP/dt and an elevation of LVEDP (Table 1). Taken together, changes in the level of IL-17A can have profound reciprocal effects on myocardial I/R injury, in terms of both infarct area and cardiac function.

IL-17A knockout ameliorates myocardial I/R injury

To elucidate the impact of IL-17A on myocardial I/R injury in the genetically IL-17A free situation, we compared myocardial I/R injury levels between Il17a–/– and wild-type C57BL/6 mice. Before surgery, we excluded the possibility of cardiac abnormality in Il17a–/– mice by echocardiography. Echocardiographic parameters of Il17a–/– mice were comparable to those of wild-type mice (Online Table 3). The Il17a–/– and wild-type mice were then subjected to myocardial I/R. The extent of injury and heart functions were assayed 1 day after initiation of reperfusion. With similar AAR, the infarct size/AAR was significantly smaller in Il17a–/– than in wild-type mice (Figs. 2A and 2B). Less serious injury was also manifested by lower levels of serum cTnT in Il17a–/– mice (Online Fig. 3B). In parallel, we observed improved cardiac function, as indicated by elevated EF and FS, in Il17a–/– mice after myocardial I/R compared with wild-type (Figs. 2C and 2D). This protection was also demonstrated by hemodynamic measurements. The LVEDP was lower, whereas LV dP/dt was higher in Il17a–/– mice at 1 day after reperfusion (Table 2). These results confirm that IL-17A plays a pathogenic role in myocardial I/R injury.

IL-17a facilitates cardiomyocyte apoptosis after I/R in vivo or oxidative stress in vitro

Apoptosis contributes significantly to myocardial I/R injury (16). We hypothesized that the role of IL-17A on myocardial I/R is associated with effects on cardiomyocyte apoptosis. To test this hypothesis, we carried out terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling (TUNEL) of LV sections from different experiment groups at 3 h after myocardial I/R. As shown in Figures 3A and 3B, anti–IL-17A mAb treatment remarkably decreased the number of TUNEL-positive cardiomyocytes compared with control, suggesting that IL17A neutralization inhibited the extent of cardiomyocyte apoptosis. In contrast, repletion of IL-17A conferred the opposite effect. Caspase 3 activity determined by a caspase colorimetric assay from ischemic myocardium was concomitantly down-regulated by anti–IL-17A mAb and up-regulated by rIL-17A (Fig. 3C). These data indicate that IL-17A has a pro-apoptotic effect on cardiomyocytes after myocardial I/R in vivo.

To test whether IL-17A directly induces cardiomyocyte apoptosis, we used primary cultures of neonatal mouse ventricular myocytes that co-expressed IL-17RA and IL-17RC, as detected by real-time polymerase chain reaction and Western blot (Online Figs. 4A and 4B). Cardiomyocytes were cultured with or without hydrogen peroxide (H2O2), a potent inducer of oxidative stress (17), in the presence or absence of rIL-17A. As expected, H2O2 treatment increased the frequency of apoptotic cardiomyocytes, whereas IL-17A incubation further augmented apoptosis. Interestingly, incubation with IL-17A alone also induced apoptosis in a dose-dependent manner (Fig. 3D). Taken together, these data indicate that IL-17A has a direct pro-apoptotic effect on cardiomyocytes.

To understand mechanisms through which IL-17A induces cardiomyocyte apoptosis, we examined expression of the Bcl-2 family by real-time polymerase chain reaction. The Bax/Bcl-2 ratio was significantly increased in cardiomyocytes exposed to H2O2. Interleukin-17A alone also increased the Bax/Bcl-2 ratio, which was further elevated under an oxidative stress situation. Administration of rIL-17 in vivo similarly increased the Bax/Bcl-2 ratio after myocardial I/R, whereas that of anti–IL-17A mAb decreased the ratio (Figs. 3E and 3F, Online Fig. 5). These results show that IL-17A induces cardiomyocyte apoptosis, at least in part, through the regulation of pro- to anti-apoptotic protein ratio of Bcl-2 family.

The CXC glutamic acid–leucine-arginine (ELR+) chemokines KC, MIP-2, and LIX are not only potent neutrophil chemoattractants but also IL-17A target genes (19). Myocardial I/R caused a significant induction of mRNA and protein levels of all 3 chemokines, consistent with increased neutrophil infiltration. Moreover, neutralization or repletion of IL-17A had the opposite effect on expression of these chemokines after myocardial I/R (Fig. 4D, Online Fig. 6A). Interleukin-17A also increased expression of cardiomyocyte of these 3 chemokines, in vitro as determined by enzyme-linked immunoadsorbent assay. Stimulation with IL-17A in addition to H2O2 led to a dose-dependent potentiation of the effect of H2O2, markedly up-regulating expression of these chemokines. Pre-incubation with anti–IL-17A Ab could completely abolish IL-17A activity (Fig. 4E, Online Fig. 6B). Furthermore, in vitro neutrophil migration assay showed a marked enhancement of neutrophil migration to media from cells treated with IL-17A or H2O2 alone and was synergistically enhanced by conditioned media from cells treated with both agents (Fig. 4F). The activity of IL-17A could be completely abolished by anti–IL-17A mAb but not isotype mAb, indicating an IL-17A–specific effect.

Adhesion molecule expression, an indicator of endothelium activation, is a prerequisite for neutrophil-endothelium adherence, which in turn contributes to myocardial I/R injury (20). To further explore the mechanisms by which IL-17A affects myocardial I/R injury, we focused on inter-cellular adhesion molecule (ICAM)-1 and E-selectin expression. After 30 min of ischemia and 3 h of reperfusion, we detected a substantial increase of ICAM-1 mRNA level and a moderate but significant increase of E-selectin mRNA levels within I/R myocardium. Treatment with anti–IL-17A mAb attenuated this induction, whereas rIL-17A treatment had the opposite effect (Fig. 5A). This effect of IL-17A on the expression of ICAM-1 and E-selectin was further confirmed by Western blotting (Online Fig. 6C). As to the cellular level: stimulation with IL-17A led to a higher surface expression of ICAM-1 and E-selectin on endothelial cells (ECs), as detected by cell-based enzyme-linked immunoadsorbent assay and an increased adherence of neutrophils to ECs as detected by neutrophil-EC adhesion assays. Moreover, addition of IL-17A to H2O2 led to a potentiation of the increased ICAM-1 and E-selectin surface expression and neutrophil adherence caused by H2O2 alone. When anti–IL-17A mAb was added, this augmentation could be absolutely blocked (Figs. 5B to 5D, Online Fig. 6D).

Discussion

The present study reveals a critical role for IL-17A in mediating mouse myocardial I/R injury. Myocardial infiltrated γδ T lymphocytes but not Th17 cells were a major source of IL-17A. Anti–IL-17A mAb treatment or IL-17A knockout markedly ameliorated I/R injury, which was associated with a reduction in cardiomyocyte apoptosis and neutrophil infiltration. Repletion of exogenous IL-17A induced the opposite effect. In vitro experiments demonstrated that IL-17A could induce cardiomyocyte apoptosis through regulation of the Bax/Bcl-2 ratio. Moreover, IL-17A enhanced neutrophil infiltration by promoting EC E-selectin and ICAM-1 expression and inducing CXC chemokine-mediated neutrophil migration.

Ischemia-reperfusion triggers a vigorous inflammatory response, augmented by the generation and release of various cytokines, chemokines, and adhesion molecules, which ultimately exacerbates tissue injury (21). Increasing evidence indicates that the elements of both adaptive immunity and innate immunity participate in I/R injury (22). Notably, IL-17A acts as a bridge between adaptive and innate immunity through the potent induction of a gene expression program typical of the inflammatory response, presenting a unique position in the immune response process (3). This finding has prompted new insight into the role of IL-17A in I/R injury. A growing body of evidence demonstrates that IL-17A is involved in the immune response during tissue I/R injury in brain, kidney, and intestine (13–15). However, little was previously known about myocardial I/R, although IL-17A is involved in the pathogenesis of diverse cardiovascular diseases (7–12). Most importantly, we have found elevated circulating levels of IL-17A in patients with acute myocardial infarction (23). Therefore, we focused on IL-17A in myocardial I/R with a mouse model. Here, we observed elevated IL-17A in the myocardium beginning at 1 h after I/R, peaking at 24 h, and decreasing thereafter, which suggests that IL17A acts in the early stages of myocardial I/R.

Although prior studies of IL-17A have focused largely on CD4+ Th17 cells, several reports indicate that γδT cells are also a major source of this cytokine (4). In our study, most of the IL-17A+ cells were CD3+ T lymphocytes. To our surprise, approximately 70% of the IL-17A–producing T lymphocytes were CD4- but TCRγδ+. The differentiation of Th17 cells from naïve-CD4 cells usually takes several days in vivo and is currently thought to depend on TCR engagement. However, IL-17–producing γδT cells can immediately respond to stimuli—such as pathogens or IL-1/IL-23—with no need for prior stimulation via the TCR and produce high amounts of IL-17A within hours (24). It is no wonder, considering the nearly instantaneous elevation of IL-17A in myocardium after I/R, that γδT lymphocytes but not Th17 cells were a major source of IL-17A, which is consistent with cerebral I/R (13). However, IL-17A–producing cells detected in kidneys after 3 h of reperfusion were CD11b+GR-1+ neutrophils (14), which differs from our results that little of IL-17A–producing cells are neutrophils. In intestinal I/R, although Edgerton et al. (15) focused on CD4+ T cells, they only examined the co-localization of CD3 and IL-17A but performed no further examination of T cell subsets. This discrepancy might be attributed to differences between tissues of I/R model.

The Il17a–/– mice showed a significant reduction in infarct volume after cerebral I/R (13). The Il17a–/– and Il17r–/– mice also showed protective effects on kidney function and morphology after renal I/R. Such protection was also observed by treatment with neutralizing anti–IL-17A mAb (14). In the present study, both neutralization of IL-17A just before induction of reperfusion and IL-17A genetic deficiency markedly ameliorated I/R injury, as demonstrated by reduced infarct size, cTnT levels, and improved cardiac function, whereas exogenous IL-17A significantly exacerbated I/R injury, demonstrating a critical role for IL-17A in myocardial I/R injury.

Apoptosis has been proposed to be an important mechanism for a significant amount of cell death in reperfused ischemic myocardium (16). It could be regulated by oxygen free radicals, cytokines such as TNF-α and IL-6, and neutrophil accumulation (25). The Bcl-2 family consists of pro- and anti-apoptotic members. The balance between pro-apoptotic and anti-apoptotic proteins determines the possibility of cells to either survive or undergo apoptosis after a certain stimulus or injury. IL-17A has been shown to induce vascular smooth muscle cell and airway epithelial cell apoptosis, but it has no effect on keratinocyte and osteoblast cell apoptosis (9,26–28). Our in vivo study showed that neutralization or repletion of IL-17A could regulate cardiomyocyte apoptosis, as confirmed by the change of TUNEL positive cardiomyocytes, caspase-3 activity, and the ratio of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins. Interleukin-17A might trigger apoptosis directly in cardiomyocytes or indirectly as a mediator that orchestrates other factors. Our in vitro study further confirmed that IL-17A had direct pro-apoptosis effect on cardiomyocyte. When the cardiomyocytes are exposed to oxidative stress, extrinsic and intrinsic signal pathways are activated, Fas mRNA and Bcl-2 family proteins are up-regulated, and the redox state is changed, which can ultimately drive cardiomyocyte into apoptosis (25,29,30). The regulation of caspase-3 activity and ratio of Bax/Bcl-2 demonstrated that IL-17A activated intrinsic signal pathways. Remarkably, myocardial IL-17R expression was not affected by I/R in vivo or oxidative stress in vitro (data not shown), therefore the mechanisms through which IL-17A alone or interaction with oxidative stress modifies the apoptosis signal pathway in myocardial I/R remain to be elucidated.

Neutrophil recruitment plays a major role in myocardial damage after I/R (18). Neutrophil chemotaxis and activation might be strongly regulated by CXC chemokines, particularly ELR+ chemokines with the tripeptide ELR motif (31). Interestingly, IL-17A specifically induced the expression of all ELR+CXCL chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8) in cultured human synoviocytes but had no effect on the expression of non-ELR CXC chemokines (CXCL9, CXCL10, and CXCL11) (32). Interleukin-17A has also been shown to induce KC, MIP-2, and LIX, which are rodent homologs of CXCL1, CXCL2, and CXCL5, in mouse osteoblastic cells (33) and lung fibroblasts (34). Moreover, the importance of ELR+CXC chemokines in myocardial I/R has been implicated in vivo (35). Here, we found that inhibition of IL-17A markedly decreased cardiac LIX, KC, and MIP-2 expression and neutrophil infiltration in mouse I/R myocardium, whereas exogenous IL-17A increased LIX, KC, and MIP-2 expression and neutrophil infiltration in mouse I/R myocardium. In vitro experiments demonstrated that IL-17A could enhance cardiomyocyte LIX, KC, and MIP-2 production and neutrophil migration to the supernatant from conditioned cardiomyocytes. Taken together, these results indicate that IL-17A contributes to myocardial I/R injury at least partially through the regulation of neutrophil infiltration to the myocardium via CXC chemokine expression.

The neutrophil-endothelium interaction is critical for neutrophil-mediated inflammation. The molecular interactions required for neutrophils to cross the endothelium are regulated via the expression of adhesion molecules on the endothelium (18). E-selectin is a key molecule for rolling, whereas ICAM-1 is important in adhesion. Deficiency in either ICAM-1 or E-selectin expression results in a marked reduction in neutrophil accumulation and myocardial injury after I/R (36). IL-17A was reported to be a potential upstream inducer of ICAM-1 and E-selectin in lung microvascular ECs (37) and human umbilical vein ECs (19). We found that anti–IL-17A mAb and rIL-17A treatments regulate the expression of ICAM-1 and E-selectin in I/R myocardium at both mRNA and protein levels. In vitro studies showed that stimulation with IL-17A resulted in endothelial activation as revealed by elevated surface expression of E-selectin and ICAM-1 as well as neutrophil-endothelial adhesion. These results, along with the neutrophil migration data, support the notion that IL-17A has a potent effect on neutrophil recruitment and adherence, which are critical processes in myocardial I/R injury.

Conclusions

We have presented evidence that IL-17A produced primarily by γδT cells plays a pathogenic role in myocardial I/R injury by inducing cardiomyocyte apoptosis and neutrophil infiltration. These data suggest a novel IL-17A-dependent pathway by which the immune system might influence the myocardial I/R injury. Control of IL-17A production might be of benefit for minimizing I/R-instigated myocardial damage.

Acknowledgments

The authors thank Prof. Qing K. Wang and Prof. Xin Tu (both at the College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, China) for assistance in echocardiography.

Appendix

For supplementary figures, tables, and Methods, please see the online version of this article.

Appendix

Footnotes

This work was supported by grants from the National Basic Research Program of China (973 Program: 2007CB512005 and 2012CB517805 to Drs. Liao and Cheng); the National Natural Science Foundation of China (no. 81170303 and 30871067 to Dr. Cheng); Program for New Century Excellent Talents in University of China (NCET-09-0380 to Dr. Cheng); and the National Institutes of Health Grants HL60942, HL81090, and HL88547 to Dr. Shi). All authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Liao, Xia, Zhou, and Tang contributed equally to this work.

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